Bacterial cystitis is overwhelmingly a syndrome of women and the kidney or urologic disease prompting the most visits to physician offices in the US. That the pathogenesis of this disease is poorly understood is ironic. It is the intention of this new project to direct the tools of molecular biology at pertinent E. coli strains in order to examine mechanisms of pathogenesis using human bladder epthelial cells (HBEC) in culture and an in vivo animal model of UTI modified to minimize kidney involvement. The majority of cystitis strains do not have P fimbriae, S fimbriae, hemolysin, and other E. coli characteristics identified as virulence factors for pyelonephritis. Therefore, we have developed an experimental plan that slows the study not only of those genes modestly epidemiologically associated with cystitis but also the identification, by functional roles, of heretofore unknown genes. Preliminary studies demonstrate the E. coli hemolysin, a pore-forming cytotoxin is active against HBEC, that HBEC internalize E. coli, and that mouse UTI virulence is not explained solely by known E. coli virulence factors. We will test the following hypotheses: 1) That strains causing cystitis in humans adhere to HBEC via specific known or as yet unrecognized adhesins. This will be demonstrated by mutagenesis of pertinent strains: isogenic for known adhesins and transposon to identify unknown adhesins. 2) That F54, a potent E. coli cystitis strain with no known virulence factors, causes cystitis in part through internalization into bladder epithelial cells. This will be approached via transposon mutagenesis to seek mutants with diminished internalization and virulence. 3) That the activities of E. coli hemolysin and cytotoxic necrotizing factor (CNF) in the living bacterium can be dissociated by genetic manipulation. Hemolysin is epidemiologically linked to cystitis and CNF is an interesting protein identified in 2/3 of hemolytic strains which enhances bacterial internalization and causes multinucleation of host epithelial cells. Our intent will be to dissociate hemolysin and CNF via isogenic mutagenesis and to study the four permutations of these two putative virulence factors in adherence, internalization, and cytotoxicity assays in order to generate hypotheses of the action and interaction of hemolysin and CNF.